Humanized anti-CDCP1 antibodies

ABSTRACT

The present invention relates to humanized antibodies against human CDCP1 (anti-CDCP1 antibody), methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/868,845, filed Aug. 25, 2010, the contents of which are herebyincorporated by reference, and which claims the benefit of the filingdate of European patent application 09011046.1, filed Aug. 28, 2009, andEuropean patent application 10000972.9, filed Feb. 1, 2010.

FIELD OF INVENTION

The present invention relates to humanized antibodies against humanCDCP1 (anti-CDCP1 antibody), methods for their production,pharmaceutical compositions containing said antibodies, and usesthereof.

BACKGROUND OF THE INVENTION

Human CDCP1 ((CUB domain containing protein 1, B345, CD318, SIMA135,TRASK; SEQ ID NO:29 and variants with mutation R525Q (i.e. replacementof Arginine (R) with Glutamine (Q) at amino acid position 525 of SEQ IDNO:29) and/or mutation G709D (i.e. replacement of Glycine (G) withAspartic acid (D) at amino acid position 709 of SEQ ID NO:29)) is atransmembrane protein containing three extracellular CUB domains. Thisprotein is found to be overexpressed in breast, colon and lung cancers.Its expression level is correlated with the metastatic ability ofcarcinoma cells (Uekita, T. et al., Am. J. Pathol. 172 (2008)1729-1739). It has been shown to be tyrosine phosphorylated in a cancercell line (WO 2002/004508; Scherl-Mostageer, M., et al., Oncogene 20(2001) 4402-8; Hooper, J., D., et al., Oncogene 22 (2003) 1783-94;Perry, S., E., et al FEBS Lett. 581 (2007) 1137-42; Brown, T., A., at alJ. Biol. Chem. 279 (2004) 14772-14783; Ota, T., et al., Nat. Genet. 36(2004) 40-45). Alternatively spliced transcript variants encodingdistinct isoforms have been reported.

WO 2002/004508 refers to CDCP1 as tumor associated antigen B345. WO2004/074481 relates to CDCP1 as glycoprotein antigen SIMA135 expressedin metastatic tumor cells. WO 2005/042102 relates to CDCP1 as proteininvolved in ovarian cancer. WO 2007/005502 relates to methods andcompositions for treating diseases targeting CDCP1.

US 2004/0053343 (and Conze, T., et al., Ann. N. Y. Acad. Sci. 996 (2003)222-6 and Buehring, H. J. et al., Stem Cells 22 (2004) 334-43) relatesto CDCP1 antibodies for identifying certain stem cell populations.

SUMMARY OF THE INVENTION

Unless specifically indicated otherwise, all amino acid positionsreferenced in this specification are numbered according to Kabat.

One aspect of the present invention is an antibody specifically bindingto human CDCP1 comprising a variable heavy chain domain (VH) of SEQ IDNO:1 and a variable light chain domain (VL) of SEQ ID NO:2 of CUB4antibody (Deposition No. DSM ACC2551),

-   -   characterized in being humanized and comprising in said VH        sequence:    -   a Lysine (K) at position 57 instead of Threonine (T), and a        Valine (V) at position 60 instead of a Proline (P).

Another aspect of the present invention is an antibody specificallybinding to human CDCP1 comprising a variable heavy chain domain (VH) ofSEQ ID NO:1 and a variable light chain domain (VL) of SEQ ID NO:2 ofCUB4 antibody (Deposition No. DSM ACC2551),

-   -   characterized in being humanized and comprising in said VL        sequence:    -   a Leucine (L) at position 33 instead of Valine (V), and a        Tryptophan (W) at position 47;

Another aspect of the present invention is an antibody specificallybinding to human CDCP1 comprising a variable heavy chain domain (VH) ofSEQ ID NO:1 and a variable light chain domain (VL) of SEQ ID NO:2 ofCUB4 antibody (Deposition No. DSM ACC2551),

-   -   characterized in being humanized,    -   and comprising in said VH sequence:    -   a Lysine (K) at position 57 instead of Threonine (T), and a        Valine (V) at position 60 instead of a Proline (P);    -   and comprising in said VL sequence:    -   a Leucine (L) at position 33 instead of Valine (V), and a        Tryptophan (W) at position 47.

Preferably the humanized antibody according to the invention ischaracterized in that the heavy chain variable domain (VH) is SEQ IDNO:3.

Preferably the humanized antibody according to the invention ischaracterized in that the light chain variable domain (VL) is SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

Preferably the humanized antibody according to the invention ischaracterized in that

-   -   the heavy chain variable domain (VH) is SEQ ID NO:3.    -   and    -   the light chain variable domain (VL) is SEQ ID NO: 14, SEQ ID        NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:        19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO:        24.

Preferably the humanized antibody according to the invention ischaracterized in that

-   -   the heavy chain variable domain (VH) is SEQ ID NO:3.    -   and    -   the light chain variable domain (VL) is SEQ ID NO: 14, SEQ ID        NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

Preferably the humanized antibody according to the invention ischaracterized in that said antibody is of human IgG1 subclass.

Preferably the humanized antibody according to the invention ischaracterized in that said antibody is glycosylated with a sugar chainat Asn297 whereby the amount of fucose within said sugar chain is 65% orlower.

A further embodiment of the invention is a pharmaceutical compositioncomprising the humanized antibody according to the invention.

A further embodiment of the invention is said pharmaceutical compositioncomprising the humanized antibody according to the invention fortreatment of cancer.

The invention further comprises the humanized antibody according to theinvention for treatment of cancer.

The invention further comprises the use the humanized antibody accordingto the invention for the preparation of a medicament for treatment ofcancer.

The invention provides nucleic acid encoding the humanized antibodyaccording to the invention. The invention further provides expressionvectors containing nucleic acid according to the invention capable ofexpressing said nucleic acid in a prokaryotic or eukaryotic host cell,and host cells containing such vectors for the recombinant production ofan antibody according to the invention.

The invention further comprises a prokaryotic or eukaryotic host cellcomprising a vector according to the invention.

The invention further comprises a method for the production of arecombinant humanized antibody according to the invention, characterizedby expressing a nucleic acid according to the invention in a prokaryoticor eukaryotic host cell and recovering said antibody from said cell orthe cell culture supernatant. The invention further comprises theantibody obtained by such a recombinant method.

The invention further provides a method for treating a patient sufferingfrom cancer, comprising administering to a patient diagnosed as havingsuch a disease (and therefore being in need of such a therapy) aneffective amount of an antibody according to the invention. The antibodyis administered preferably in a pharmaceutical composition.

It has now surprisingly found out that the specific humanized versionsof the CDCP1 antibody CUB4 according the invention show improvedCDCP1-binding properties compared to other humanized versionsoriginating from humanizations known in the prior art. This is due tospecific amino acid changes in the CDRH2, and/or in the CDRL1 and in theframework of the light chain. Surprisingly the specific humanizedversions of the CDCP1 antibody CUB4 according the invention showimproved in vivo tumor growth inhibition compared to the chimeric andmouse CUB4 antibodies.

DESCRIPTION OF THE FIGURES

FIG. 1 VH domain amino acid sequence (CDRH1, CDRH2 and CDRH3 aremarked—bold letters) of mouse (mVH-CUB4) antibody and VH domain aminoacid sequences of different humanized CUB4 anti-CDCP1 antibodies(specific modifications according to the invention are marked—boldletters)

FIG. 2 VL domain amino acid sequence (CDRL1, CDRL2 and CDRL3 aremarked—bold letters) of mouse (mVL-CUB4) antibody and VL domain aminoacid sequences of different humanized CUB4 anti-CDCP1 antibodies(specific modifications according to the invention are marked—boldletters)

FIG. 3 Relative binding ratios of different humanized anti-CDCP1antibodies CUB4. The binding ratios of the combinations of differenthumanized VH and VL domain relative to chimeric CUB4 ((chHC4=mouse VHand VL with human IgG1 constant region) antibody is shown.

FIG. 4 in vitro ADCC of glycoengineered (GE) humanized CUB4 antibody No.69 GE with an amount of fucose of 65% or less compared with the ADCC ofthe chimeric glycoengineered (GE) CUB4 and wildtype (wt=nonglycoengineered chimeric CUB4 Antibody and negative human IgG control.

FIGS. 5a and 5b In vivo tumor growth inhibition in human lung cancerH322M xenograft of humanized CUB4 Antibodies No. 69 and No. 135,chimeric CUB4 antibody and mouse CUB4 antibody.

DETAILED DESCRIPTION OF THE INVENTION

The CUB4 antibody refers to the deposited antibody with the DepositionNo. DSM ACC2551 (DSMZ) from DE10242146 (EP 1 396 501, U.S. Pat. No.7,541,030) with the heavy chain variable domain (VH) of SEQ ID NO:1 andthe light chain variable domain (VL) of SEQ ID NO:2. Said CUB4 antibodyis specifically binding to human CDCP1.

The term “being humanized” as used herein denotes an antibody, based onthe deposited mouse CUB4 antibody with the VH of SEQ ID NO:1 and the VLof SEQ ID NO:2, in which (after chimerization with a human constantregion) said VH and VL are humanized by grafting the murine CDRs intothe framework region of a human antibody (see e.g. Riechmann, L., etal., Nature 332 (1988) 323-327; and Neuberger, M., S., et al., Nature314 (1985) 268-270; Queen, C., et al., Proc. Natl. Acad. Sci. USA 86(1989) 10029-10033; U.S. Pat. No. 5,530,101; U.S. Pat. No. 5,585,089;U.S. Pat. No. 5,693,761; WO 90/07861; and U.S. Pat. No. 5,225,539). Theheavy and light chain variable framework regions can be derived from thesame or different human antibody sequences. The human antibody sequencescan be the sequences of naturally occurring human antibodies. Humanheavy and light chain variable framework regions are listed e.g. inLefranc, M. P., Current Protocols in Immunology (2000)—Appendix 1PA.1P.1-A.1P.37 and are accessible via IMGT, the internationalImMunoGeneTics information System® (http://imgt.cines.fr) or viahttp://vbase.mrc-cpe.cam.ac.uk.

The humanized antibodies according to the invention have in addition

-   -   a) specific mutations in the CDRH2 of the VH (mutations T57K and        P60V). and/or    -   b) specific mutations in CDRL1 of the VL (mutation V33L) and in        the framework region of VL (backmutation from human VL framework        amino acid to mouse amino acid W at position 47).

Such mutations in the humanized CUB antibodies surprisingly lead toimproved binding properties (compared to humanized CUB4 antibodieswithout such modifications). Furthermore such modifications in the CDRsand/or the framework resulted in the humanized antibodies according tothe invention with improved in vivo tumor growth inhibition (compared tothe chimeric and mouse parent antibodies).

One aspect of the present invention is an antibody specifically bindingto human CDCP1 comprising a variable heavy chain domain (VH) of SEQ IDNO:1 and a variable light chain domain (VL) of SEQ ID NO:2 of CUB4antibody (Deposition No. DSM ACC2551),

characterized in being humanized and comprising in said VH sequence:

a Lysine (K) at position 57 instead of Threonine (T) (in the CDRH2), anda Valine (V) at position 60 instead of a Proline (P) (in the CDRH2) (allpositions being numbered according to Kabat). This means that SEQ IDNO:1 comprises the mutations T57K and P60V in the CDRH2 of VH.

Another aspect of the present invention is an antibody specificallybinding to human CDCP1 comprising a variable heavy chain domain (VH) ofSEQ ID NO:1 and a variable light chain domain (VL) of SEQ ID NO:2 ofCUB4 antibody (Deposition No. DSM ACC2551),

characterized in being humanized and comprising in said VL sequence:

a Leucine (L) at position 33 instead of Valine (V) (in the CDRL1), and aTryptophan (W) at position 47 (instead of an amino acid from a human VLframework region) (all positions being numbered according to Kabat).This means that SEQ ID NO:2 comprise the mutation V33L in the CDRL1 anda backmutation from human to mouse amino acid W at position 47 in theframework region VL.

Another aspect of the present invention is an antibody specificallybinding to human CDCP1 comprising a variable heavy chain domain (VH) ofSEQ ID NO:1 and a variable light chain domain (VL) of SEQ ID NO:2 ofCUB4 antibody (Deposition No. DSM ACC2551),

characterized in being humanized,

and comprising in said VH sequence:

a Lysine (K) at position 57 instead of Threonine (T) (in the CDRH2), anda Valine (V) at position 60 instead of a Proline (P) (in the CDRH2); andcomprising in said VL sequence:

a Leucine (L) at position 33 instead of Valine (V) and a Tryptophan (W)at position 47 (all positions being numbered according to Kabat). Thismeans that SEQ ID NO:1 comprises the mutations T57K and P60V in theCDRH2 of VH and that SEQ ID NO:2 comprises the mutation V33L in theCDRL1 of VL and a backmutation from human to mouse amino acid W atposition 47 in the framework region of VL.

Another aspect of the present invention is an antibody specificallybinding to human CDCP1 comprising a variable heavy chain domain (VH) ofSEQ ID NO:1 and a variable light chain domain (VL) of SEQ ID NO:2 ofCUB4 antibody (Deposition No. DSM ACC2551),

characterized in being humanized and comprising in said VL sequence:

a Leucine (L) at position 33 instead of Valine (V) (in the CDRL1), and aTryptophan (W) at position 47 (instead of an amino acid from a human VLframework region);

and being further characterized in (further) comprising in said VLsequence:

a Methionine (M) at position 21 (instead of an amino acid from a humanVL framework region) (all positions being numbered according to Kabat).

Another aspect of the present invention is an antibody specificallybinding to human CDCP1 comprising a variable heavy chain domain (VH) ofSEQ ID NO:1 and a variable light chain domain (VL) of SEQ ID NO:2 ofCUB4 antibody (Deposition No. DSM ACC2551),

characterized in being humanized,

and comprising in said VH sequence:

a Lysine (K) at position 57 instead of Threonine (T) (in the CDRH2), anda Valine (V) at position 60 instead of a Proline (P) (in the CDRH2);

and comprising in said VL sequence:

a Leucine (L) at position 33 instead of Valine (V) and a Tryptophan (W)at position 47 (instead of an amino acid from a human VL frameworkregion);

and being further characterized in (further) comprising in said VLsequence:

a Methionine (M) at position 21 (instead of an amino acid from a humanVL framework region) (all positions being numbered according to Kabat).

Another aspect of the present invention is an antibody specificallybinding to human CDCP1 comprising a variable heavy chain domain (VH) ofSEQ ID NO:1 and a variable light chain domain (VL) of SEQ ID NO:2 ofCUB4 antibody (Deposition No. DSM ACC2551),

characterized in being humanized and comprising in said VL sequence:

a Leucine (L) at position 33 instead of Valine (V) (in the CDRL1), and aTryptophan (W) at position 47 (instead of an amino acid from a human VLframework region);

and being further characterized in (further) comprising in said VLsequence:

a Methionine (M) at position 21 (instead of an amino acid from a humanVL framework region); a Glycine (G) or a Arginine (R) at position 24instead of a Serine (S) (in the CDRL1), and Alanine (A) at position 25instead of a Valine (V) (in the CDRL1) (all positions being numberedaccording to Kabat).

Another aspect of the present invention is an antibody specificallybinding to human CDCP1 comprising a variable heavy chain domain (VH) ofSEQ ID NO:1 and a variable light chain domain (VL) of SEQ ID NO:2 ofCUB4 antibody (Deposition No. DSM ACC2551),

characterized in being humanized,

and comprising in said VH sequence:

a Lysine (K) at position 57 instead of Threonine (T) (in the CDRH2), anda Valine (V) at position 60 instead of a Proline (P) (in the CDRH2);

and comprising in said VL sequence:

a Leucine (L) at position 33 instead of Valine (V) and a Tryptophan (W)at position 47 (instead of an amino acid from a human VL frameworkregion;

and being further characterized in (further) comprising in said VLsequence:

a Methionine (M) at position 21 (instead of an amino acid from a humanVL framework region), a Glycine (G) or a Arginine (R) at position 24instead of a Serine (S) (in the CDRL1), and Alanine (A) at position 25instead of a Valine (V) (in the CDRL1) (all positions being numberedaccording to Kabat).

Another aspect of the present invention is an antibody specificallybinding to human CDCP1 comprising a variable heavy chain domain (VH) ofSEQ ID NO:1 and a variable light chain domain (VL) of SEQ ID NO:2 ofCUB4 antibody (Deposition No. DSM ACC2551),

characterized in being humanized and comprising in said VL sequence:

a Leucine (L) at position 33 instead of Valine (V) (in the CDRL1), and aTryptophan (W) at position 47 (instead of an amino acid from a human VLframework region);

and being further characterized in (further) comprising in said VLsequence:

a Arginine (R) at position 24 instead of a Serine (S) (in the CDRL1),and Alanine (A) at position 25 instead of a Valine (V) (in the CDRL1)(all positions being numbered according to Kabat).

Another aspect of the present invention is an antibody specificallybinding to human CDCP1 comprising a variable heavy chain domain (VH) ofSEQ ID NO:1 and a variable light chain domain (VL) of SEQ ID NO:2 ofCUB4 antibody (Deposition No. DSM ACC2551),

characterized in being humanized,

and comprising in said VH sequence:

a Lysine (K) at position 57 instead of Threonine (T) (in the CDRH2), anda Valine (V) at position 60 instead of a Proline (P) (in the CDRH2);

and comprising in said VL sequence:

a Leucine (L) at position 33 instead of Valine (V) and a Tryptophan (W)at position 47 (instead of an amino acid from a human VL frameworkregion;

and being further characterized in (further) comprising in said VLsequence:

a Arginine (R) at position 24 instead of a Serine (S) (in the CDRL1),and Alanine (A) at position 25 instead of a Valine (V) (in the CDRL1)(all positions being numbered according to Kabat).

In one embodiment of the invention the humanized antibody according tothe invention is characterized in that

-   -   the heavy chain variable domain (VH) is SEQ ID NO:3.

In another embodiment of the invention the humanized antibody accordingto the invention is characterized in that

-   -   the light chain variable domain (VL) is SEQ ID NO: 14, SEQ ID        NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

In another embodiment of the invention the humanized antibody accordingto the invention is characterized in that

-   -   the heavy chain variable domain (VH) is SEQ ID NO:3.    -   and    -   the light chain variable domain (VL) is SEQ ID NO: 14, SEQ ID        NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:        19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO:        24.

In another embodiment of the invention the humanized antibody accordingto the invention is characterized in that

-   -   the heavy chain variable domain (VH) is SEQ ID NO:3.    -   and    -   the light chain variable domain (VL) is SEQ ID NO: 14, SEQ ID        NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

In another embodiment of the invention the humanized antibody accordingto the invention is characterized in that

-   -   the heavy chain variable domain (VH) is SEQ ID NO:3.    -   and    -   the light chain variable domain (VL) is SEQ ID NO: 14.

In another embodiment of the invention the humanized antibody accordingto the invention is characterized in that

-   -   the heavy chain variable domain (VH) is SEQ ID NO:3.    -   and    -   the light chain variable domain (VL) is SEQ ID NO: 15.

In another embodiment of the invention the humanized antibody accordingto the invention is characterized in that

-   -   the heavy chain variable domain (VH) is SEQ ID NO:3.    -   and    -   the light chain variable domain (VL) is SEQ ID NO: 16.

In another embodiment of the invention the humanized antibody accordingto the invention is characterized in that

-   -   the heavy chain variable domain (VH) is SEQ ID NO:3.    -   and    -   the light chain variable domain (VL) is SEQ ID NO: 17.

In another embodiment of the invention the humanized antibody accordingto the invention is characterized in that

-   -   the heavy chain variable domain (VH) is SEQ ID NO:3.    -   and    -   the light chain variable domain (VL) is SEQ ID NO: 18.

In another embodiment of the invention the humanized antibody accordingto the invention is characterized in that

-   -   the heavy chain variable domain (VH) is SEQ ID NO:3.    -   and    -   the light chain variable domain (VL) is SEQ ID NO: 23.

In another embodiment of the invention the humanized antibody accordingto the invention is characterized in that said antibody is of human IgG1subclass.

In another embodiment of the invention the humanized antibody accordingto the invention is characterized in that said antibody is glycosylatedwith a sugar chain at Asn297 whereby the amount of fucose within saidsugar chain is 65% or lower.

Preferred embodiments of the humanized antibody according to theinvention are characterized by one of the following combinations of ahumanized variable heavy chain domain VH and a humanized variable lightchain domain VL, as shown in Table 1 (see the following Example Nos.).

TABLE 1 Preferred combinations of a humanized variable heavy chaindomain VH and a humanized variable light chain domain VL. Humanized CUB4Antibody Example No VL (SEQ ID NO:) VL (SEQ ID NO:) 80 hHC4-H (SEQ IDNO: 3) hLC-M (SEQ ID NO: 14) 69 hHC4-H (SEQ ID NO: 3) hLC-L2 (SEQ ID NO:15) 47 hHC4-H (SEQ ID NO: 3) hLC-K (SEQ ID NO: 16) 58 hHC4-H (SEQ ID NO:3) hLC-L (SEQ ID NO: 17) 36 hHC4-H (SEQ ID NO: 3) hLC-J (SEQ ID NO: 18)102 hHC4-H (SEQ ID NO: 3) hLC-b (SEQ ID NO: 19) 113 hHC4-H (SEQ ID NO:3) hLC-c (SEQ ID NO: 20) 91 hHC4-H (SEQ ID NO: 3) hLC-a (SEQ ID NO: 21)124 hHC4-H (SEQ ID NO: 3) hLC-d (SEQ ID NO: 22) 135 hHC4-H (SEQ ID NO:3) hLC-e (SEQ ID NO: 23) 146 hHC4-H (SEQ ID NO: 3) hLC-f (SEQ ID NO: 24)

Further preferred embodiments of the humanized antibody according to theinvention are characterized by one of the following combinations of ahumanized variable heavy chain domain VH and a humanized variable lightchain domain VL, as shown in Table 2. Such combinations comprising ahuman the following Example No., as shown in Table 2.

TABLE 2 Further preferred combinations of a humanized variable heavychain domain VH and a humanized variable light chain domain VL.Humanized CUB4 Antibody Example No VL (SEQ ID NO:) VL (SEQ ID NO:) 80hHC4-H (SEQ ID NO: 3) hLC-M (SEQ ID NO: 14) 69 hHC4-H (SEQ ID NO: 3)hLC-L2 (SEQ ID NO: 15) 47 hHC4-H (SEQ ID NO: 3) hLC-K (SEQ ID NO: 16) 58hHC4-H (SEQ ID NO: 3) hLC-L (SEQ ID NO: 17) 36 hHC4-H (SEQ ID NO: 3)hLC-J (SEQ ID NO: 18)

The term “Kabat numbering” or “numbering according to Kabat” or “EUindex” unless otherwise stated, is defined as the numbering of theresidues in, e.g., an IgG antibody using the EU index as in Kabat et al.(Sequences of Proteins of Immunological Interest, 5th ed., Public HealthService, National Institutes of Health, Bethesda, Md. (1991)).

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of a singleamino acid composition.

The term “chimeric antibody” refers to a monoclonal antibody comprisinga variable region, i.e., binding region, from mouse and at least aportion of a constant region derived from a different source or species,usually prepared by recombinant DNA techniques. Chimeric antibodiescomprising a mouse variable region and a human constant region areespecially preferred. Such mouse/human chimeric antibodies are theproduct of expressed immunoglobulin genes comprising DNA segmentsencoding mouse immunoglobulin variable regions and DNA segments encodinghuman immunoglobulin constant regions. Other forms of “chimericantibodies” encompassed by the present invention are those in which theclass or subclass has been modified or changed from that of the originalantibody. Such “chimeric” antibodies are also referred to as“class-switched antibodies.” Methods for producing chimeric antibodiesinvolve conventional recombinant DNA and gene transfection techniquesnow well known in the art (see, e.g., Morrison, S., L., et al., Proc.Natl. Acad Sci. USA 81 (1984) 6851-6855; U.S. Pat. No. 5,202,238 andU.S. Pat. No. 5,204,244).

Human CDCP1 ((CUB domain containing protein 1, B345, CD318, SIMA135,TRASK; SEQ ID NO:29 and variants with mutation R525Q (i.e. replacementof Arginine (R) with Glutamine (Q) at amino acid position 525 of SEQ IDNO:29) and/or mutation G709D (i.e. replacement of Glycine (G) withAspartic acid (D) at amino acid position 709 of SEQ ID NO:29)) is atransmembrane protein containing three extracellular CUB domains. Thisprotein is found to be overexpressed in breast, colon and lung cancers(Uekita, T. et al., Am. J. Pathol. 172 (2008) 1729-1739). Its expressionlevel is correlated with the metastatic ability of carcinoma cells. Ithas been shown to be tyrosine phosphorylated in a cancer cell line (WO2002/004508; Scherl-Mostageer, M., et al., Oncogene 20 (2001) 4402-8;Hooper, J., D., et al., Oncogene 22 (2003) 1783-94; Perry, S. E., etal., FEBS Lett. 581 (2007) 1137-42; Brown, T. A., et al., J. Biol. Chem.279 (2004) 14772-14783; Ota, T., et al., Nat. Genet. 36 (2004) 40-45).Alternatively spliced transcript variants encoding distinct isoformshave been reported.

As used herein, “specifically binding to human CDCP1” refers to anantibody specifically binding to the human CDCP1 antigen. The bindingaffinity is of KD-value of 1.0×10⁻⁸ mol/l or lower (e.g. 1.0×10⁻⁸ mol/lto 1.0×10⁻¹³ mol/l), preferably of a KD-value of 5.0×10⁻⁹ mol/l or lower(e.g. 5.0×10⁻⁹ mol/l to 1.0×10⁻¹³ mol/l). The binding affinity isdetermined with a standard binding assay, such as surface plasmonresonance technique (Biacore®).

The term “epitope” denotes a protein determinant of human CDCP1 capableof specifically binding to an antibody. Epitopes usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually epitopes have specific three dimensionalstructural characteristics, as well as specific charge characteristics.Conformational and nonconformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents.

The “variable domain” (variable domain of a light chain (VL), variabledomain of a heavy chain (VH)) as used herein denotes each of the pair oflight and heavy chain domains which are involved directly in binding theantibody to the antigen. The variable light and heavy chain domains havethe same general structure and each domain comprises four framework (FR)regions whose sequences are widely conserved, connected by three“hypervariable regions” (or complementary determining regions, CDRs).The framework regions adopt a β-sheet conformation and the CDRs may formloops connecting the β-sheet structure. The CDRs in each chain are heldin their three-dimensional structure by the framework regions and formtogether with the CDRs from the other chain the antigen binding site.The antibody's heavy and light chain CDR3 regions play a particularlyimportant role in the binding specificity/affinity of the antibodiesaccording to the invention and therefore provide a further object of theinvention.

“Framework” or “FR” regions are those variable domain regions other thanthe hypervariable region residues as herein defined. Therefore, thelight and heavy chain variable domains of an antibody comprise from N-to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRand FR regions are determined according to the standard definition ofKabat et al., Sequences of Proteins of Immunological Interest, 5th ed.,Public Health Service, National Institutes of Health, Bethesda, Md.(1991) and/or those residues from a “hypervariable loop”.

The term “antigen-binding portion of an antibody” when used herein referto the amino acid residues of an antibody which are responsible forantigen-binding. The antigen-binding portion of an antibody comprisesamino acid residues from the “complementary determining regions” or“CDRs”. The term “antigen-binding portion” of an antibody of theinvention contains six complementarity determining regions (CDRs) whichcontribute in varying degrees to the affinity of the binding site forantigen. There are three heavy chain variable domain CDRs (CDRH1, CDRH2and CDRH3) and three light chain variable domain CDRs (CDRL1, CDRL2 andCDRL3). The term “CDRH1” denotes the CDR1 region of the heavy chainvariable region calculated according to Kabat. CDRH2, CDRH3, CDRL1,CDRL2 and CDRL3 mean the respective regions from the heavy (H) or light(L) chain. The extent of CDR and framework regions (FRs) is determinedby comparison to a compiled database of amino acid sequences in whichthose regions have been defined according to variability among thesequences according to Kabat, et al., supra.

The terms “nucleic acid” or “nucleic acid molecule”, as used herein, areintended to include DNA molecules and RNA molecules. A nucleic acidmolecule may be single-stranded or double-stranded, but preferably isdouble-stranded DNA.

The term “amino acid” as used within this application denotes the groupof naturally occurring carboxy α-amino acids comprising alanine (threeletter code: ala, one letter code: A), arginine (arg, R), asparagine(asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q),glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine(ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M),phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine(thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

The antibody according to the invention is characterized in that theconstant region is of human origin, and is preferably of human IgG1subclass. The constant region includes the heavy chain and light chainconstant region of an antibody. The heavy chain constant regioncomprises in N-terminal to C-terminal direction an antibody constantheavy chain domain 1 (CH1), an antibody hinge region (HR), an antibodyheavy chain constant domain 2 (CH2), and an antibody heavy chainconstant domain 3 (CH3), and optionally, in case of an antibody of thesubclass IgE, an antibody heavy chain constant domain 4 (CH4). The lightchain constant region comprises an antibody light chain constant domain(CL). The antibody light chain constant domain (CL) can be κ (kappa) orλ (lambda). Such constant chains are well known in the state of the artand e.g. described by Kabat, E. A., (see e.g. Johnson, G. and Wu, T.,T., Nucleic Acids Res. 28 (2000) 214-218). For example, a useful humanheavy chain constant region of IgG1 subclass comprises an amino acidsequence of SEQ ID NO: 26. For example, a useful human light chainconstant region comprises an amino acid sequence of a kappa-light chainconstant region of SEQ ID NO: 27; another useful human light chainconstant region comprises an amino acid sequence of a lambda-light chainconstant region of SEQ ID NO: 28.

The “Fc part” of an antibody is not involved directly in binding of anantibody to an antigen, but exhibit various effector functions. A “Fcpart of an antibody” is a term well known to the skilled artisan anddefined on the basis of papain cleavage of antibodies. Depending on theamino acid sequence of the constant region of their heavy chains,antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. Accordingto the heavy chain constant regions the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively.

The Fc part of an antibody is directly involved in ADCC(antibody-dependent cell-mediated cytotoxicity) and CDC(complement-dependent cytotoxicity) based on complement activation, C1qbinding and Fc receptor binding. Complement activation (CDC) isinitiated by binding of complement factor C1q to the Fc part of most IgGantibody subclasses. While the influence of an antibody on thecomplement system is dependent on certain conditions, binding to C1q iscaused by defined binding sites in the Fc part. Such binding sites areknown in the state of the art and described e.g. by Boackle, R. J., etal., Nature 282 (1979) 742-743; Lukas, T. J., et al., J. Immunol. 127(1981) 2555-2560; Brunhouse, R., and Cebra, J. J., Mol. Immunol. 16(1979) 907-917; Burton, D. R., et al., Nature 288 (1980) 338-344;Thommesen, J. E., et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, E.E., et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J.Virology 75 (2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995)319-324; EP 0307434. Such binding sites are e.g. L234, L235, D270, N297,E318, K320, K322, P331 and P329 (numbering according to EU index ofKabat, E. A., see below). Antibodies of subclass IgG1, IgG2 and IgG3usually show complement activation and C1q and C3 binding, whereas IgG4do not activate the complement system and do not bind C1q and C3.

The antibody according to the invention comprises a Fc part derived fromhuman origin and preferably all other parts of the human constantregions. As used herein the term “Fc part derived from human origin”denotes a Fc part which is either a Fc part of a human antibody of thesubclass IgG1, IgG2, IgG3 or IgG4, preferably a Fc part from human IgG1subclass, a mutated Fc part from human IgG1 subclass (preferably with amutation on L234A+L235A), a Fc part from human IgG4 subclass or amutated Fc part from human IgG4 subclass (preferably with a mutation onS228P). Mostly preferred are the human heavy chain constant regions ofhuman IgG1 subclass with SEQ ID NO: 26 or 31, of human IgG1 subclasswith mutations L234A and L235A, of human IgG4 subclass with SEQ ID NO:32, or of human IgG4 subclass with mutation S228P.

The term “antibody-dependent cellular cytotoxicity (ADCC)” refers tolysis of human target cells by an antibody according to the invention inthe presence of effector cells. ADCC is measured preferably by thetreatment of a preparation of CDCP1 expressing cells with an antibodyaccording to the invention in the presence of effector cells such asfreshly isolated PBMC or purified effector cells from buffy coats, likemonocytes or natural killer (NK) cells or a permanently growing NK cellline.

The term “complement-dependent cytotoxicity (CDC)” denotes a processinitiated by binding of complement factor C1q to the Fc part of most IgGantibody subclasses. Binding of C1q to an antibody is caused by definedprotein-protein interactions at the so called binding site. Such Fc partbinding sites are known in the state of the art (see above). Such Fcpart binding sites are, e.g., characterized by the amino acids L234,L235, D270, N297, E318, K320, K322, P331, and P329 (numbering accordingto EU index of Kabat). Antibodies of subclass IgG1, IgG2, and IgG3usually show complement activation including C1q and C3 binding, whereasIgG4 does not activate the complement system and does not bind C1qand/or C3.

Cell-mediated effector functions of monoclonal antibodies can beenhanced by engineering their oligosaccharide component as described inUmana, P., et al., Nature Biotechnol. 17 (1999) 176-180, and U.S. Pat.No. 6,602,684. IgG1 type antibodies, the most commonly used therapeuticantibodies, are glycoproteins that have a conserved N-linkedglycosylation site at Asn297 in each CH2 domain. The two complexbiantennary oligosaccharides attached to Asn297 are buried between theCH2 domains, forming extensive contacts with the polypeptide backbone,and their presence is essential for the antibody to mediate effectorfunctions such as antibody dependent cellular cytotoxicity (ADCC)(Lifely, M., R., et al., Glycobiology 5 (1995) 813-822; Jefferis, R., etal., Immunol. Rev. 163 (1998) 59-76; Wright, A., and Morrison, S., L.,Trends Biotechnol. 15 (1997) 26-32). Umana, P., et al., NatureBiotechnol. 17 (1999) 176-180 and WO 99/54342 showed that overexpressionin Chinese hamster ovary (CHO) cells ofβ(1,4)-N-acetylglucosaminyltransferase III (“GnTIII”), aglycosyltransferase catalyzing the formation of bisectedoligosaccharides, significantly increases the in vitro ADCC activity ofantibodies. Alterations in the composition of the Asn297 carbohydrate orits elimination affect also binding to FcγR and C1q (Umana, P., et al.,Nature Biotechnol. 17 (1999) 176-180; Davies, J., et al., Biotechnol.Bioeng. 74 (2001) 288-294; Mimura, Y., et al., J. Biol. Chem. 276 (2001)45539-45547; Radaev, S., et al., J. Biol. Chem. 276 (2001) 16478-16483;Shields, R. L., et al., J. Biol. Chem. 276 (2001) 6591-6604; Shields, R.L., et al., J. Biol. Chem. 277 (2002) 26733-26740; Simmons, L. C., etal., J. Immunol. Methods 263 (2002) 133-147).

Methods to enhance cell-mediated effector functions of monoclonalantibodies are reported e.g. in WO 2005/044859, WO 2004/065540,WO2007/031875, Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180,WO 99/154342, WO 2005/018572, WO 2006/116260, WO 2006/114700, WO2004/065540, WO 2005/011735, WO 2005/027966, WO 1997/028267, US2006/0134709, US 2005/0054048, US 2005/0152894, WO 2003/035835 and WO2000/061739 or e.g. in Niwa, R., et al., J. Immunol. Methods 306 (2005)151-160; Shinkawa, T., et al., J. Biol. Chem. 278 (2003) 3466-3473; WO03/055993 and US 2005/0249722.

Therefore in one embodiment of the invention, the antibody according tothe invention is glycosylated (if it comprises an Fc part of IgG1 orIgG3 subclass) with a sugar chain at Asn297 whereby the amount of fucosewithin said sugar chain is 65% or lower (Numbering according to Kabat).In another embodiment is the amount of fucose within said sugar chain isbetween 5% and 65%, preferably between 20% and 40%. “Asn297” accordingto the invention means amino acid asparagine located at about position297 in the Fc region. Based on minor sequence variations of antibodies,Asn297 can also be located some amino acids (usually not more than ±3amino acids) upstream or downstream of position 297, i.e. betweenposition 294 and 300. In one embodiment the glycosylated antibodyaccording to the invention the IgG subclass is of human IgG1 subclass,or of IgG3 subclass. In a further embodiment the amount ofN-glycolylneuraminic acid (NGNA) is 1% or less and/or the amount ofN-terminal alpha-1,3-galactose is 1% or less within said sugar chain.The sugar chains show preferably the characteristics of N-linked glycansattached to Asn297 of an antibody recombinantly expressed in a CHO cell.

The term “the sugar chains show characteristics of N-linked glycansattached to Asn297 of an antibody recombinantly expressed in a CHO cell”denotes that the sugar chain at Asn297 of the antibody according to theinvention has the same structure and sugar residue sequence except forthe fucose residue as those of the same antibody expressed in unmodifiedCHO cells, e.g. as those reported in WO 2006/103100.

The term “NGNA” as used within this application denotes the sugarresidue N-glycolylneuraminic acid.

Glycosylation of human IgG1 or IgG3 occurs at Asn297 as core fucosylatedbiantennary complex oligosaccharide glycosylation terminated with up totwo Gal residues. Human constant heavy chain regions of the IgG1 or IgG3subclass are reported in detail by Kabat, E., A., et al., supra, and byBrueggemann, M., et al., J. Exp. Med. 166 (1987) 1351-1361; Love, T. W.,et al., Methods Enzymol. 178 (1989) 515-527. These structures aredesignated as G0, G1 (α-1,6- or α-1,3-), or G2 glycan residues,depending from the amount of terminal Gal residues (Raju, T., S.,Bioprocess Int. 1 (2003) 44-53). CHO type glycosylation of antibody Fcparts is e.g. described by Routier, F. H., Glycoconjugate J. 14 (1997)201-207. Antibodies which are recombinantly expressed innon-glycomodified CHO host cells usually are fucosylated at Asn297 in anamount of at least 85%. The modified oligosaccharides of the antibodymay be hybrid or complex. Preferably the bisected,reduced/not-fucosylated oligosaccharides are hybrid. In anotherembodiment, the bisected, reduced/not-fucosylated oligosaccharides arecomplex.

According to the invention “amount of fucose” means the amount of saidsugar within the sugar chain at Asn297, related to the sum of allglycostructures attached to Asn297 (e.g. complex, hybrid and highmannose structures) measured by MALDI-TOF mass spectrometry andcalculated as average value (see e.g. WO 2008/077546). The relativeamount of fucose is the percentage of fucose-containing structuresrelated to all glycostructures identified in an N-Glycosidase F treatedsample (e.g. complex, hybrid and oligo- and high-mannose structures,resp.) by MALDI-TOF.

The antibodies according to the invention are preferably produced byrecombinant means. Such methods are widely known in the state of the artand comprise protein expression in prokaryotic and eukaryotic cells withsubsequent isolation of the antibody polypeptide and usuallypurification to a pharmaceutically acceptable purity. For the proteinexpression, nucleic acids encoding light and heavy chains or fragmentsthereof are inserted into expression vectors by standard methods.Expression is performed in appropriate prokaryotic or eukaryotic hostcells like CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells,yeast, or E. coli cells, and the antibody is recovered from the cells(supernatant or cells after lysis).

Recombinant production of antibodies is well-known in the state of theart and described, for example, in the review articles of Makrides, S.,C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., ProteinExpr. Purif. 8 (1996) 271-282; Kaufman, R. J., Mol. Biotechnol. 16(2000) 151-160; Werner, R. G., Drug Res. 48 (1998) 870-880.

The antibodies may be present in whole cells, in a cell lysate, or in apartially purified or substantially pure form. Purification is performedin order to eliminate other cellular components or other contaminants,e.g. other cellular nucleic acids or proteins, by standard techniques,including alkaline/SDS treatment, CsCl banding, column chromatography,agarose gel electrophoresis, and others well known in the art (seeAusubel, F., et al. (ed.), Current Protocols in Molecular Biology,Greene Publishing and Wiley Interscience, New York (1987).

Expression in NS0 cells is described by, e.g., Barnes, L. M., et al.,Cytotechnology 32 (2000) 109-123; and Barnes, L. M., et al., Biotech.Bioeng. 73 (2001) 261-270. Transient expression is described by, e.g.,Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning ofvariable domains is described by Orlandi, R., et al., Proc. Natl. Acad.Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods204 (1997) 77-87. A preferred transient expression system (HEK 293) isdescribed by Schlaeger, E. J., and Christensen, K., in Cytotechnology 30(1999) 71-83 and by Schlaeger, E. J., in J. Immunol. Methods 194 (1996)191-199.

The control sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters, enhancersand polyadenylation signals.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading frame. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The monoclonal antibodies are suitably separated from the culture mediumby conventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography. DNA and RNAencoding the monoclonal antibodies are readily isolated and sequencedusing conventional procedures. The hybridoma cells can serve as a sourceof such DNA and RNA. Once isolated, the DNA may be inserted intoexpression vectors, which are then transfected into host cells such asHEK 293 cells, CHO cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of recombinantmonoclonal antibodies in the host cells.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

The term “transformation” as used herein refers to process of transferof a vectors/nucleic acid into a host cell. If cells without formidablecell wall barriers are used as host cells, transfection is carried oute.g. by the calcium phosphate precipitation method as described byGraham, F., L., and van der Eb, Virology 52 (1973) 456-467. However,other methods for introducing DNA into cells such as by nuclearinjection or by protoplast fusion may also be used. If prokaryotic cellsor cells which contain substantial cell wall constructions are used,e.g. one method of transfection is calcium treatment using calciumchloride as described by Cohen, S. N., et al., PNAS 69 (1972) 2110-2114.

As used herein, “expression” refers to the process by which a nucleicacid is transcribed into mRNA and/or to the process by which thetranscribed mRNA (also referred to as transcript) is subsequently beingtranslated into peptides, polypeptides, or proteins. The transcripts andthe encoded polypeptides are collectively referred to as gene product.If the polynucleotide is derived from genomic DNA, expression in aeukaryotic cell may include splicing of the mRNA.

A “vector” is a nucleic acid molecule, in particular self-replicating,which transfers an inserted nucleic acid molecule into and/or betweenhost cells. The term includes vectors that function primarily forinsertion of DNA or RNA into a cell (e.g., chromosomal integration),replication of vectors that function primarily for the replication ofDNA or RNA, and expression vectors that function for transcriptionand/or translation of the DNA or RNA. Also included are vectors thatprovide more than one of the functions as described.

An “expression vector” is a polynucleotide which, when introduced intoan appropriate host cell, can be transcribed and translated into apolypeptide. An “expression system” usually refers to a suitable hostcell comprised of an expression vector that can function to yield adesired expression product.

One aspect of the invention is a pharmaceutical composition comprisingan antibody according to the invention. Another aspect of the inventionis the use of an antibody according to the invention for the manufactureof a pharmaceutical composition. A further aspect of the invention is amethod for the manufacture of a pharmaceutical composition comprising anantibody according to the invention. In another aspect, the presentinvention provides a composition, e.g. a pharmaceutical composition,containing an antibody according to the present invention, formulatedtogether with a pharmaceutical carrier.

Furthermore such specific humanized versions of the CDCP1 antibody CUB4have turned out to be especially useful for the treatment of cancercompared with e.g. other anti-CDCP1 antibodies

Therefore one aspect of the invention is said pharmaceutical compositionfor the treatment of cancer.

Another aspect of the invention is the humanized antibody according tothe invention for the treatment of cancer.

Another aspect of the invention is the use of the humanized antibodyaccording to the invention for the manufacture of a medicament for thetreatment of cancer.

Another aspect of the invention is a method of treatment of a patientsuffering from cancer by administering the humanized antibody accordingto the invention to said patient in the need of such treatment.

As used herein, “pharmaceutical carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g. by injection or infusion).

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. To administer a compound of the invention bycertain routes of administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation. For example, the compound may be administered to asubject in an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Pharmaceutical carriers include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intra-arterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

The term “cancer” as used herein may be, for example, lung cancer, nonsmall cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bonecancer, pancreatic cancer, skin cancer, cancer of the head or neck,cutaneous or intraocular melanoma, uterine cancer, ovarian cancer,rectal cancer, cancer of the anal region, stomach cancer, gastriccancer, colon cancer, breast cancer, uterine cancer, carcinoma of thefallopian tubes, carcinoma of the endometrium, carcinoma of the cervix,carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease,cancer of the esophagus, cancer of the small intestine, cancer of theendocrine system, cancer of the thyroid gland, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, prostate cancer, cancer of thebladder, cancer of the kidney or ureter, renal cell carcinoma, carcinomaof the renal pelvis, mesothelioma, hepatocellular cancer, biliarycancer, neoplasms of the central nervous system (CNS), spinal axistumors, brain stem glioma, glioblastoma multiforme, astrocytomas,schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cellcarcinomas, pituitary adenoma, lymphoma, lymphocytic leukemia, includingrefractory versions of any of the above cancers, or a combination of oneor more of the above cancers. Preferably such cancer is a breast cancer,ovarian cancer, cervical cancer, lung cancer or prostate cancer and morepreferably lung cancer. Preferably such cancers are furthercharacterized by CDCP1 expression or overexpression, more preferably byCDCP1 overexpression

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol, sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carrierpreferably is an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by use of coating suchas lecithin, by maintenance of required particle size in the case ofdispersion and by use of surfactants. In many cases, it is preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol or sorbitol, and sodium chloride in the composition.

The following examples, sequence listing and figures are provided to aidthe understanding of the present invention, the true scope of which isset forth in the appended claims. It is understood that modificationscan be made in the procedures set forth without departing from thespirit of the invention.

DESCRIPTION OF THE AMINO ACID SEQUENCES OF THE SEQUENCE LISTING

-   -   SEQ ID NO:1 heavy chain variable domain VH of CUB4 (Deposition        No. DSM ACC2551)    -   SEQ ID NO:2 light chain variable domain VL of CUB4 (Deposition        No. DSM ACC2551)    -   SEQ ID NO:3 hHC4-H-humanized VH of CUB4    -   SEQ ID NO:4 hHC4-c-humanized VH of CUB4    -   SEQ ID NO:5 hHC4-a-humanized VH of CUB4    -   SEQ ID NO:6 hHC4-d-humanized VH of CUB4    -   SEQ ID NO:7 hHC4-04-humanized VH of CUB4    -   SEQ ID NO:8 hHC4-K-humanized VH of CUB4    -   SEQ ID NO:9 hHC4-K2-humanized VH of CUB4    -   SEQ ID NO:10 hHC4-I-humanized VH of CUB4    -   SEQ ID NO:11 hHC4-07-humanized VH of CUB4    -   SEQ ID NO:12 hHC4-03-humanized VH of CUB4    -   SEQ ID NO:13 hHC4-b-humanized VH of CUB4    -   SEQ ID NO:14 hLC4-M-humanized VL of CUB4    -   SEQ ID NO:15 hLC4-L2-humanized VL of CUB4    -   SEQ ID NO:16 hLC4-K-humanized VL of CUB4    -   SEQ ID NO:17 hLC4-L-humanized VL of CUB4    -   SEQ ID NO:18 hLC4-J-humanized VL of CUB4    -   SEQ ID NO:19 hLC4-b-humanized VL of CUB4    -   SEQ ID NO:20 hLC4-c-humanized VL of CUB4    -   SEQ ID NO:21 hLC4-a-humanized VL of CUB4    -   SEQ ID NO:22 hLC4-d-humanized VL of CUB4    -   SEQ ID NO:23 hLC4-e-humanized VL of CUB4    -   SEQ ID NO:24 hLC4-f-humanized VL of CUB4    -   SEQ ID NO:25 hLC4-I-humanized VL of CUB4    -   SEQ ID NO:26 IgG1 constant heavy chain region from human origin        (Caucasian Allotype)    -   SEQ ID NO:27 kappa constant light chain region from human origin    -   SEQ ID NO:28 lambda constant light chain region from human        origin    -   SEQ ID NO:29 human CDCP1    -   SEQ ID NO:30 extracellular-domain-(ECD)-comprising fragment of        human CDCP1    -   SEQ ID NO:31 IgG1 constant heavy chain region from human origin        (Afroamerican Allotype)    -   SEQ ID NO:32 IgG4 constant heavy chain region from human origin

EXAMPLE 1 Antigene Specific ELISA

Soluble CDCP1 extracellular domain (CDCP1-ECD) (SEQ ID NO:30) fused toStreptavidin Binding Protein (SBP) was captured on a sreptavidine plate.To define optimal binding of the antibody to SBP-CDCP1-ECD, 384-wellpolystyrene plates (NUNC, streptavidin-coated) delivered by MicroCoat,Bernried, Germany (ID-No. 1734776-001) have been coated with pure andstepwise diluted HEK293 supernatant (in BSA/IMDM buffer:100 mg/ml BSAFraction V, Roche 10735078001, dissolved in Iscove's Modified DulbeccosMedium). Using a calibration curve of mouse CUB4 antibodies the optimaldilution factor of the HEK293 supernatant in relation to thestreptavidin binding capacity of the microtiter plate was identified.For the standard coating, SBP-CDCP1-ECD containing HEK293 supernatantwas diluted (between 1:15 and 1:40) and incubated overnight at 2-8° C.(25 μl per well). Intensive washing of the microtiter plate is necessaryto remove remaining unbound SBP-CDCP1-ECD.

Humanized CUB4 antibodies and/or reference antibody (chimeric (chHC4)CUB4 antibody comprising human constant region and mouse VH and VL ofSEQ ID NO:1 and 2) were tested either undiluted or using a12-step-dilution. 12.5 μl per well of each sample was incubated for 90min at room temperature. After intensive washing using PBS-T (0.1% Tween20 in PBS) 25 μl of either goat anti-human IgG antibodies coupled withHRP (Jackson ImmunoResearch, Code No: 109-036-098, dilution 1:10000) forhuman antibodies were added and incubated for 1 hour. After intensivewashing the binding of the antibodies was detected with ABTS tablets(Roche Diagnostics GmbH, Cat. No.: 1112422). Absorbance at 405 nm/492 nmwas measured using a standard photometer.

In FIG. 3 the binding ratios of the different combinations of humanizedVH and VL relative to chimeric (chHC4) CUB4 antibody is shown. Theresults show that the specific modified humanized antibodies with

-   -   a) specific mutations in the CDRH2 of the VH (mutations T57K and        P60V). (see VH domain: hHC4-H (SEQ ID NO: 3),    -   and/or    -   b) specific mutations in CDRL1 of the VL (mutation V33L) and a        mutation from human to mouse amino acid W at position 47 in the        VL framework region; (see VL domains: hLC4-M (SEQ ID NO: 14),        hLC4-L2 (SEQ ID NO: 15), hLC4-K (SEQ ID NO: 16), hLC4-L (SEQ ID        NO: 17), hLC4-J (SEQ ID NO: 18))

surprisingly lead to clearly improved binding properties compared tohumanized CUB4 antibodies without such specific modifications.

EXAMPLE 2 Characterization of the Binding of Anti-CDCP1 Antibodies to aExtracellular-Domain-(ECD)-Comprising Fragment of Human CDCP1 of SEQ IDNO.: 30 (Comprising the Extracellular Domain ECD of Human CDCP1)

For affinity measurements, 30 μg/ml anti mouse Fcγ antibodies (fromgoat, Jackson Immuno Research JIR115-005-071) were coupled to thesurface of a CM-5 sensor chip by standard amine-coupling and blockingchemistry on a SPR instrument (Biacore T100). After conjugation,different anti-CDCP1 antibodies were injected at 25° C. at a flow rateof 5 μL/min, followed by a dilution series (0 nM to 1000 nM) of CDCP1ECD at 30 μL/min. As running buffer for the binding experiment PBS/0.1%BSA was used. The chip was then regenerated with a 60 s pulse of 10 mMglycine-HCl, pH 2.0 solution.

Calculation of thermodynamic parameters (K_(D), binding constant) andkinetic parameters (k_(on) rate, k_(off) rate) were calculated using aLangmuir 1:1 binding model.

TABLE 3 Exemplary binding parameters of humanized antibodies accordingto the invention Humanized Anti- k_(a) k_(d) t_(1/2) diss. K_(D) CDCP1antibody [1/Ms] [1/s] [min] [nM] 47 2.0E+05 7.5E−04 15.4 3.77 69 2.1E+055.2E−04 22.1 2.55 80 2.5E+05 8.8E−04 13.1 3.53 91 1.8E+05 1.0E−03 11.25.60 102 1.9E+05 1.1E−03 11.0 5.40 135 1.4E+05 1.1E−03 10.3 8.30

EXAMPLE 3 Preparation of Glycoengineered Humanized CUB4 Antibody(Humanized CUB4 Antibody GE)

The full antibody heavy and light chain DNA sequences corresponding tothe amino acid sequences SEQ ID NO:3 and SEQ ID NO:15 (Antibody 69) weresubcloned into mammalian expression vectors (one for the light chain andone for the heavy chain) under the control of the MPSV promoter andupstream of a synthetic polyA site, each vector carrying an EBV OriPsequence.

Antibodies were produced by co-transfecting HEK293-EBNA cells with themammalian antibody heavy and light chain expression vectors using acalcium phosphate-transfection approach. Exponentially growingHEK293-EBNA cells were transfected by the calcium phosphate method. Forthe production of unmodified antibody, the cells were transfected onlywith antibody heavy and light chain expression vectors in a 1:1 ratio.For the production of the glycoengineered antibody, the cells wereco-transfected with four plasmids, two for antibody expression, one fora fusion GnTIII polypeptide expression (a GnT-III expression vector),and one for mannosidase II expression (a Golgi mannosidase II expressionvector) at a ratio of 4:4:1:1, respectively. Cells were grown asadherent monolayer cultures in T flasks using DMEM culture mediumsupplemented with 10% FCS, and were transfected when they were between50 and 80% confluent. For the transfection of a T150 flask, 15 millioncells were seeded 24 hours before transfection in 25 ml DMEM culturemedium supplemented with FCS (at 10% V/V final), and cells were placedat 37° C. in an incubator with a 5% CO2 atmosphere overnight. For eachT150 flask to be transfected, a solution of DNA, CaCl2 and water wasprepared by mixing 94 μg total plasmid vector DNA divided equallybetween the light and heavy chain expression vectors, water to a finalvolume of 469 μl and 469 μl of a 1M CaCl2 solution. To this solution,938 μl of a 50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4 solution at pH 7.05were added, mixed immediately for 10 sec and left to stand at roomtemperature for 20 sec. The suspension was diluted with 10 ml of DMEMsupplemented with 2% FCS, and added to the T150 in place of the existingmedium. Then additional 13 ml of transfection medium were added. Thecells were incubated at 37° C., 5% CO2 for about 17 to 20 hours, thenmedium was replaced with 25 ml DMEM, 10% FCS. The conditioned culturemedium was harvested 7 days post-transfection by centrifugation for 15min at 210×g, the solution was sterile filtered (0.22 μm filter) andsodium azide in a final concentration of 0.01% w/v was added, and keptat 4° C.

The secreted antibody glycoengineered humanized CUB4 Antibodies No. 69(humanized CUB4 Antibodies No. 69 GE) was purified by Protein A affinitychromatography, followed by cation exchange chromatography and a finalsize exclusion chromatographic step on a Superdex 200 column (AmershamPharmacia) exchanging the buffer to 25 mM potassium phosphate, 125 mMsodium chloride, 100 mM glycine solution of pH 6.7 and collecting thepure monomeric IgG1 antibodies. Antibody concentration was estimatedusing a spectrophotometer from the absorbance at 280 nm. Theoligosaccharides attached to the Fc region of the antibodies wereanalysed by MALDI/TOF-MS (as described in e.g. WO 2008/077546).Oligosaccharides were enzymatically released from the antibodies byPNGaseF digestion, with the antibodies being either immobilized on aPVDF membrane or in solution. The resulting digest solution containingthe released oligosaccharides either prepared directly for MALDI/TOF-MSanalysis or was further digested with EndoH glycosidase prior to samplepreparation for MALDI/TOF-MS analysis.

In a further experiment the glycoengineered humanized CUB4 Antibodies(humanized CUB4 Antibody GE) Antibody 69 and 135 were prepared byco-transfection with four plasmids, two for antibody expression, one fora fusion GnTIII polypeptide expression (a GnT-III expression vector),and one for mannosidase II expression (a Golgi mannosidase II expressionvector) at a ratio of 4:4:1:1, respectively in CHO cells instead ofHEK293-EBNA cells. The analyzed amount of fucose within the sugar chainat Asn297 was between 50-10%.

EXAMPLE 4 In Vitro ADCC of Humanized CUB4 Antibodies

The target cells PC-3 (DSMZ #ACC 465, prostatic adenocarcinoma,cultivation in Ham's F12 Nutrient Mixture+2 mM L-alanyl-L-Glutamine+10%FCS) and H322M (non small cell lung carcinoma, cultivation in RPMI1640+2mM L-alanyl-L-Glutamine+10% FCS) were collected with trypsin/EDTA (Gibco#25300-054) in exponential growth phase. After a washing step andchecking cell number and viability the aliquot needed was labeled for 30min at 37° C. in the cell incubator with calcein (Invitrogen #C3100MP; 1vial was resuspended in 50 μl DMSO for 5 Mio cells in 5 ml medium).Afterwards, the cells were washed three times with AIM-V medium, thecell number and viability was checked and the cell number adjusted to0.3 Mio/ml.

Meanwhile, PBMC as effector cells were prepared by density gradientcentrifugation (Histopaque-1077, Sigma #H8889) according to themanufacturer's protocol (washing steps 1× at 400 g and 2× at 350 g 10min each). The cell number and viability was checked and the cell numberadjusted to 15 Mio/ml.

100 μl calcein-stained target cells were plated in round-bottom 96-wellplates, 50 μl diluted antibody was added and 50 μl effector cells. Insome experiments the target cells were mixed with Redimune® NF Liquid(ZLB Behring) at a concentration of 10 mg/ml Redimune.

As controls served the spontaneous lysis, determined by co-culturingtarget and effector cells without antibody and the maximal lysis,determined by 1% Triton X-100 lysis of target cells only. The plate wasincubated for 4 hours at 37° C. in a humidified cell incubator.

The killing of target cells was assessed by measuring LDH release fromdamaged cells using the Cytotoxicity Detection kit (LDH Detection Kit,Roche #1 644 793) according to the manufacturer's instruction. Briefly,100 μl supernatant from each well was mixed with 100 μl substrate fromthe kit in a transparent flat bottom 96 well plate. The Vmax values ofthe substrate's colour reaction was determined in an ELISA reader at 490nm for at least 10 min. Percentage of specific antibody-mediated killingwas calculated as follows: ((A−SR)/(MR−SR)×100, where A is the mean ofVmax at a specific antibody concentration, SR is the mean of Vmax of thespontaneous release and MR is the mean of Vmax of the maximal release.

As additional readout the calcein retention of intact target cells wasassessed by lysing the remaining target cells in borate buffer (5 mMsodium borate+0.1% Triton) and measuring the calcein fluorescence in afluorescence plate reader.

FIG. 4 shows the in vitro ADCC of glycoengineered (GE) humanized CUB4Antibody No. 69 GE with an amount of fucose of 65% or less compared withthe ADCC of the chimeric glycoengineered (GE) CUB4 and wildtype (wt=nonglycoengineered chimeric CUB4 Antibody and negative human IgG control.

EXAMPLE 5 Stimulation of CDCP1 Phosphorylation in DU-145 Cells

2×10⁵ per 6 well Du-145 cells cultured in DMEM (Paa cat. No. E15-0011) 2mM L-glutamine (Sigma Cat. No. G7513, 2 mM sodium pyrovate, 10% FCS (PAACat. No E15-0011) over night. Cells were incubated with 20 μg/ml of thedifferent humanized CUB4 antibodies for 10 min. Cess were lysed withfreshly prepared ice cooled RIPA lyses buffer. (RIPA—Puffer 1% NP40, 1%DOC, 0.1% SDS, 150 mM NaCl, 10 mM Tris/HCl, pH 7.4, 1 mM PMSF inEthanol, 10 μg/mL Aprotinin, 0.4 mM Orthovanadat). After 10 min on icecell lysates were centrifuged for 10 min at 10000 rpm. The lysates wereseparated on SDS-PAGe by standard protocol and transferred tonitrocellulose by Western blotting. Western Blots were detected by ananti-phosphotyrosine antibody (4G10) or an anti-phosphoCDCP1 antibody.The intensity of the phosphorylated CDCP1 was determined by densiometricscanning (Biorad GS 800 densiometer).

TABLE 4 Percentage (%) stimulation of humanized CUB4 antibodies(relative to chimeric CUB4) Humanized CUB4 Antibody No. Percent (%)stimulation Chimeric CUB4 100%  80 133%  69 112   47 96 135 72

All Humanized CUB4 Antibodies No. 80, No. 69, 47 and 135 showed astimulation of CDCP1 phosphorylation in DU-145 cells.

EXAMPLE 6 In Vivo Tumor Inhibition of Humanized CUB4 Antibodies

A) Study name: CDCP1_PZ_H322M_007

The present in vivo study was performed to compare the efficacy ofchimeric anti-CDCP1 antibody CUB4 with humanized versions of CUB4antibody in the NCI-H322M non small cell lung cancer model.

H322M non small cell lung cancer cells were obtained from the NCIcollection. Tumor cell line was routinely cultured in RPMI 1640 mediumsupplemented with 10% fetal bovine serum and 2 mM L-glutamine at 37° C.in a water-saturated atmosphere at 5% CO2. Passage 4 was used for celltransplantation.

The human non small cell lung cancer cell line H322M was subcutaneouslyinoculated (5×10⁶ cells) with matrigel into the right flank of the mice.

Animal treatment started at the day of randomisation, 19 days after celltransplantation. Antibodies were administered i.p. q7d on study day 19,26, 33, 40, and 47 at the indicated dosage of 25 mg/kg. Also thecorresponding vehicle was administered on the same days. Theadministration volume was 10 ml/kg.

Humanized CUB4 Antibody No. 69 is based on VH and VL of SEQ ID NO:3 andSEQ ID NO:15.

Humanized CUB4 Antibody No. 135 is based on VH and VL of SEQ ID NO:3 andSEQ ID NO:23.

Groups:

Treatment Group 1: Vehicle

Treatment Group 2: Chimeric CUB4 (25 mg/kg i.p).;

Treatment Group 3: Humanized CUB4 Antibody No. 69 (25 mg/kg i.p).;

Treatment Group 4: Humanized CUB4 Antibody No. 135 (25 mg/kg i.p).;

In FIG. 5a the in vivo tumor growth inhibition in human lung cancerH322M xenograft of humanized CUB4 Antibodies No. 69 and No. 135 andchimeric CUB4 antibody shown with a clearly improved in vivo tumorgrowth inhibition of both humanized CUB4 Antibodies No. 69 and No. 135compared to chimeric CUB4 antibody.

B) Study name: CDCP1_PZ_H322M_004

The present in vivo study was performed to compare the efficacy ofmurine anti-CDCP1 antibody CUB4 with chimeric anti-CDCP1 antibody CUB4(mouse VH and VL with human IgG1 constant region) in the NCI-H322M nonsmall cell lung cancer model.

H322M non small cell lung cancer cells were obtained from the NCIcollection. Tumor cell line was routinely cultured in RPMI 1640 mediumsupplemented with 10% fetal bovine serum and 2 mM L-glutamine at 37° C.in a water-saturated atmosphere at 5% CO2. Passage 4 was used for celltransplantation.

The human non small cell lung cancer cell line H322M was subcutaneouslyinoculated (5×10⁶ cells) with matrigel into the right flank of the mice.

Animal treatment started at the day of randomisation 17 days after celltransplantation. Antibodies were administered i.p. q7d until studytermination day 59 at the indicated dosage of 10 mg/kg. Also thecorresponding vehicle was administered on the same days. Theadministration volume was 10 ml/kg.

Groups:

Treatment Group 1: Vehicle

Treatment Group 4: Murine CUB4 (10 mg/kg i.p.)

Treatment Group 5: Chimeric CUB4 (10 mg/kg i.p.)

In FIG. 5b the in vivo tumor growth inhibition in human lung cancerH322M xenograft of mouse CUB4 antibody and chimeric CUB4 antibody isshown with an improved in vivo tumor growth inhibition of chimeric CUB4antibody compared to mouse CUB4 antibody.

What is claimed is:
 1. A method for treating a patient suffering from aCDCP1 expressing cancer, said method comprising administering to apatient diagnosed as having such a disease an effective amount of apharmaceutical composition comprising an antibody which specificallybinds to human CDCP1 comprising: (a) a variable heavy chain domain (VH)of SEQ ID NO:3, or (b) a variable light chain domain (VL) of SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ IDNO:24.
 2. The method of claim 1, wherein said CDCP1 expressing cancer isa breast cancer, an ovarian cancer, a cervical cancer, a lung cancer ora prostate cancer.